Chemicals or environmental factors which may increase the frequency of heritable germ cell mutations, particularly chromosome aberrations. represent significant hazards to human health. Mutagenic and genotoxic agents released into the environment also represent significant hazards to the economic well being of humans since they can have severe negative effects on the reproduction and long term survival of a variety of economically important plant and animal species.
Genomic aberrations acquired by developing and mature germ cells may contribute to sterility, fetal death, congenital abnormalities or metabolic disorders. Chemical agents and environmental agents that may induce genomic aberrations in developing gametes therefore represent significant hazards to the health and reproduction of humans and other animal species and should be screened routinely for this capacity; however, few such agents have been evaluated adequately.
A variety of in vivo and in vitro assays have been developed to evaluate mutagenic and/or genotoxic agents in somatic cells, but with few exceptions most are not adequate to assess the ability of the agents to induce chromosome aberrations in developing germ cells, especially in the male germ line. Vertebrate spermatogenesis is a complex pathway of cellular differentiation comprised of a mitotic phase of stem cell (spermotogonia) proliferation, followed by a unique premeiotic S phase, meiosis,and spermatid maturation. Due to unique characteristics of each phase, it may be expected that they will vary in their sensitivities to different mutagenic and genotoxic agents. The frequency of induction of dominant lethals and heritable translocations is indeed stage specific.
Clearly, the potential of mutagenic and genotoxic agents to induce heritable chromosome aberrations in the male germ line should be evaluated in male germ line genotoxicity and heritable mutation assays. The most appropriate testing procedures currently used are the in vivo mammalian germ cell heritable mutation assays, such as the mouse specific locus test (W. L. Russell, 1951. Cold Spring Harbor Symp. Quant. Biol. 16:327-336: L. B. Russel et al., 1981. Mutat. Res. 86:329-354). the rodent dominant lethal assay (V. H. Ehling et al., 1978, Arch. Toxicol. 39:173-185; Green et al., 1985, Mutat. Res. 154:49-67), and the heritable translocation test (Generoso et al., 1980. Mutat. Res. 76:191-215; Leonard. A. and Adler, I. D., 1984. In "Handbook of Mutagenicity Test Procedures" (Kilbey et al., eds.) Elsevier, pp. 485-494).
The specific locus and heritable translocation tests are essential for risk estimation in mammals since they are in vivo tests that measure heritable mutations which represent direct health hazards. Due to the requirements for large numbers of animals and extensive facilities however, the in vivo tests are prohibitively expensive, tedious and time consuming. For example, the fastest and least expensive mammalian heritable mutation assay (dominant lethal test) ranges in cost between $15,000 and $20.000 and requires 10 to 12 weeks for a nonduplicated test. This test is also the least sensitive in vivo assay. A mouse specific locus test may cost more than $100.000 (for a review of test costs, see D. Brusick and A. Auletta, 1985, Mutation Research 153:110).
Given the number of mutagenic and genotoxic agents and the variety of concentrations that should be tested, it is not realistic to expect routine evaluation by the heritable mutation assays. Such in vivo assays also suffer a lack of sensitivity; test agents often must be applied in doses that cause a generalized toxicity that may indirectly result in germ line genotoxicity.
Drosophila heritable mutation assays are rapid and economical, and they are capable of measuring multiple genetic endpoints (Wurgler. F. E. et al., 1984. In "Handbook of Mutagenicity Test Procedures" (B. J., Kilbey et al., eds.) Elsevier, pp. 555-601). Male meiosis in Drosophila, however, is atypical since synaptonemal complexes do not form, and homologous chromosomes do not recombine and form chiasmata. Spermatogenesis in Drosophila, moreover, is physiologically distant from that in vertebrates as evidenced by the short time (8-10 days) required for sperm development.
Another approach to screening chemicals as potential inducers of heritable mutations is to employ the less costly and more rapid in vivo mammalian germ cell genotoxicity assays. These assays include cytogenetics of spermatogonial and spermatocyte chromosomes, spermatid micronucleus assays (Lahdetie,. J., 1983 Mutat. Res. 119:79-82 and 120:257-260). unscheduled DNA synthesis (UDS) assays (Sega. G. A., 1979. Genetics 92:549-558), and alkaline elution assays for DNA breaks in spermatogenic cells (Sega. G. A. and Owens, J. G., 1982. Environ. Mutagen 4:347-348: Sega. G. A. et al., 1986, Mutat. Res. 159:55-63). Since each of these assays follows in vivo protocols, they suffer some of the drawbacks of the heritable mutation assays. These assays are also incapable of estimating the frequency of specific chromosome aberrations that appear in functional sperm and thus represent significant health hazards.
In vitro screening tests that could identify agents as candidates for further risk assessment in in vivo mammalian assays would be valuable additions to a testing program for germ cell mutagenicity and genotoxicity. Ideally, such tests would employ sperm and spermatogenic cells, permit evaluation of a wide range of test agent concentrations, be highly sensitive to a variety of test agents, and be cost effective.
Progress towards developing in vitro assays has been hindered in part by an inability to maintain spermotogenesis in vitro. Isolated mammalian spermatogenic cells rapidly degenerate in cell culture, and only limited development of mammalian spermatogonia and spermatocytes through meiotic prophase has been maintained in vitro in testis organ cultures (Steinberger and Steinberger, 1965, J. Repro. Fertil. 9:243-248: Steinberger. 1975, Hardman J. G., O'Malley B. W. (eds). Methods in Enzymology, New York: Academic Press, 283-296) and spermatogenic cell-Sertoli cell co-cultures (Tres and Kierszenbaum, 1983, Proc. Nat'l Acad. Sci. USA 80:3377-3381). Development from pachytene to round spermatid stages has been achieved recently in 7 day cultures of rat seminiferous tubules (Parvinen et al., 1983, Endocrinology 112:1150-1152; Toppari and Parvinen, 1985. J. Androl. 6:334-343) but in vitro development of mammalian spermatogenic cells beyond the round spermatid stage has not been observed.
Spermatogenic cells from amphibians and insects can be maintained in vitro more readily than their mammalian counterparts. In cell cultures, isolated spermatocytes from the frog Xenopus laevis develop from meiotic prophase to elongate spermatid stages (Risley and Eckhardt, 1979, J. Exp. Zool. 207:513-520; Risley, 1983, Gam Res. 4:331-346), and spermatocytes from the newt Cynops pyrrhogaster develop from early meiotic prophase to the round spermatid stage (Abe. 1981, Differentiation 20:65-70); Nishikawa and Abe, 1983, Develop. Growth. Differ. 25:232-331). Development from primary spermatocyte stages to late spermatid stages has also been observed in cultured testes from silkworms (Kambysellis and Williams, 1972, Science 175:769-770) and in cultured testes (Fowler GL. 1973, Cell. Differ. 2:33-42) and spermatocysts from Drosophila (Cross and Shellenbarger, 1979 J. Embryol. Exp. Morphol. 53:345-351; Liebrich, 1981, Cell Tissue Res. 220:251-262). Despite the advanced in vitro differentiation obtained with amphibian and insect spermatogenic cells, development of spermatogonia to postmeiotic stages and differentiation of sperm have not been observed in culture.
Many of the disadvantages in current screening tests could be circumvented by the development of an in vitro mutagenicity and genotoxicity test that assays most spermatogenic stages in a vertebrate. The capability to assay multiple genetic endpoints would also be advantageous. In vitro germ line screening tests have not been developed to date since an animal model for in vitro spermatogenesis was not available.